The secretion of growth factors, peptide hormones, neuropeptides and biogenic amines from dense-core vesicles (DCVs) in neurons and endocrine cells is a tightly-regulated event that drives physiological processes such as feeding, digestion, energy storage, lactation, emotion and analgesia. Compromised DCV release is implicated in metabolic and neurological disorders such as diabetes, eating disorders, depression, drug addiction, and Huntington?s disease. Yet the molecular pathways that govern the release of DCVs, particularly in electrically excitable cells of the nervous and endocrine systems, remain largely undefined. The objective of this proposal is to uncover molecular mechanisms that regulate DCV secretion. Our central hypothesis is that the signaling pathways that govern DCV release vary between different classes of cells, and between different populations of DCVs within the same cell, according to their selective expression and trafficking of key, as of yet unidentified regulatory molecules. We further posit that, similar to small synaptic vesicles, DCV release is tightly controlled by neuromodulatory signaling through G protein-coupled receptors (GPCRs). Our innovative hypothesis challenges the existing paradigm that focuses exclusively on intracellular calcium as the primary molecular determinant of DCV release. The discovery of diverse release mechanisms will provide a new understanding for long-standing questions surrounding the challenges associated with evoking neuropeptide secretion. We will test our hypothesis by addressing the following key knowledge gaps: 1) an understanding of the neural activity patterns and wide range of intracellular calcium concentrations that drive DCV release in different neuron classes, 2) and understanding of how neuromodulatory biochemical signaling can adjust the activity and/or calcium requirements for release, 3) elucidation of endogenous GPCRs that can carry out this novel form of neuromodulatory cross-talk, 4) elucidation of the diverse protein machineries associated with DCVs containing different cargoes in different cell classes. The proposed research builds on 1) our recent establishment of several assays for monitoring the actions of tachykinin and opioid neuropeptides in the striatum, 2) our recent discovery of diverse conditions for driving endogenous tachykinin and opioid neuropeptide release, 3) our successful development of photoactivatable peptides for mimicking, and thus calibrating, spatiotemporal aspects of endogenous release, and 4) the recent development of optical sensors that report peptide release in brain tissue. Uncovering the general principles that govern DCV release will establish new connections between intercellular and intracellular signaling pathways and reveal how they are integrated at the molecular level in numerous biological systems that transmit information via DCV secretion. In the long term, we anticipate that the unique signaling pathways uncovered can be exploited to treat metabolic diseases, psychological disorders and neurodegenerative disease, and for chronic pain, latter of which is urgently needed to address the Opioid Crisis. By uncovering new connections between signaling pathways that are fundamental to human physiology in both health and disease, the findings of this work will likely impact numerous scientific fields, including cancer, cardiology, development, gastroenterology, and neuroscience. 1